Hairpin ribozymes

ABSTRACT

Hairpin ribozyme lacking a substrate moiety, comprising atleast six bases in helix 2 and able to base-pair with a separate substrate RNA, wherein the said ribozyme comprises one or more bases 3&#39; of helix 3 able to base-pair with the said substrate RNA to form a helix 5 and wherein the said ribozyme can cleave and/or ligate said separate RNA(s) in trans.

This Application is a continuation of U.S. Ser. No. 08/773,297 filedDec. 23, 1996, now U.S. Pat. No. 5,837,855, which is a continuation ofU.S. Ser. No. 08/321,993, filed Oct. 11, 1994 and now U.S. Pat. No.5,631,359. The entirety of each of these applications, including thedrawings, are hereby incorporated by reference herein.

BACKGROUND OF THE INVENTION

The following is a brief description of publications concerningribozymes, and in particular, hairpin ribozymes. none are admitted to bethe prior art to the pending claims, and all are incorporated byreference herein.

Six basic varieties of naturally-occurring enzymatic RNAs are knownpresently. Each can catalyze the hydrolysis of RNA phosphodiester bondsin trans (and thus can cleave other RNA molecules) under physiologicalconditions. Table I summarizes some of the characteristics of theseribozymes. In general, enzymatic nucleic acids act by first binding to atarget RNA. Such binding occurs through the target binding portion ofenzymatic nucleic acid which is held in close proximity to an enzymaticportion of the molecule that acts to cleave the target RNA. Thus, theenzymatic nucleic acid first recognizes and then binds a target RNAthrough complementary base-pairing, and once bound to the correct site,acts enzymatically to cut the target RNA. Strategic cleavage of such atarget RNA will destroy its ability to direct synthesis of an encodedprotein. After an enzymatic nucleic acid has bound and cleaved its RNAtarget, it is released from that RNA to search for another target andcan repeatedly bind and cleave new targets.

The enzymatic nature of a ribozyme is advantageous over othertechnologies, such as antisense technology (where a nucleic acidmolecule generally simply binds to a nucleic acid target to block itstranslation) since the concentration of ribozyme necessary to affect atherapeutic treatment is lower than that of an antisenseoligonucleotide. This advantage reflects the ability to the ribozyme toact enzymatically. Thus, a single ribozyme molecule is able to cleavemany molecules of target RNA. In addition, the ribozyme is a highlyspecific inhibitor, with the specificity of inhibition depending notonly on the base pairing mechanism of binding to the target RNA, butalso on the mechanism of target RNA cleavage. Single mismatches, orbase-substitutions, near the site of cleavage can completely eliminatecatalytic activity of a ribozyme. Similar mismatches in antisensemolecules do not prevent their action (Woolf et al., 1992 Proc. Natl.Acad. Sci. USA, 89, 7305-7309). Thus, the specificity of action of aribozyme is greater than that of an antisense oligonucleotide bindingthe same RNA site.

Van Tol et al., 1991 (Virology 180, 23) describe a hairpin ribozymestructure able to circularize. Hisamatsu et al., 1993 (Nucleic AcidsSymp. Ser. 29, 173) describe hairpin ribozymes having a long substratebinding site in helix 1. Berzal-Herranz et al., 1993 (EMBO J. 12,2567)describe essential nucleotides in the hairpin ribozyme. Hampel andTritz, 1989 (Biochemistry 28, 4929) describe a hairpin ribozyme derivedfrom the minus strand of tobacco ringspot virus satellite [(-) sTRSV]RNA. Haseloff and Gerlach 1989 (Gene 82, 43) describe sequences requiredfor self-cleavage reactions catalyzed by the (-) sTRSV RNA. Feldstein etal., 1989 (Gene 82, 53) tested various models oi trans-cleaving motifsderived from (-) sTRSV RNAs. The hairpin ribozyme can be assembled invarious combinations to catalyze a unimolecular, bimolecular or atrimolecular cleavage/ligation reaction (Berzal-Herranz et al., 1992,Genes & Develop. 6, 129; Chowrira and Burke, 1992 Nucleic Acids Res. 20,2835; Komatsu et al., 1993 Nucleic Acids Res. 21, 185; Komatsu et al.,1994 J. Am. Chem. Soc, 116, 3692). Increasing the length of helix 1 andhelix 4 regions do not affect the catalytic activity of the hairpinribozyme (Hisamatsu et al., 1993 supra; Chowrira and Burke, 1992 supra;Anderson et aL, 1994 Nucleic Acids Res, 22, 1096). For a review ofvarious ribozyme motifs, and hairpin ribozyme in particular, see Ahsenand Schroeder, 1993 Bioessays 15, 299; Cech, 1992 Curr. Opi. Struc. Bio.2, 605; and Hampel et aL, 1993 Methods: A Companion to Methods inEnzymology 5, 37.

This invention concerns an improved ribozyme based on the hairpin motifdescribed by Hampel and Fritz 1989 supra; Feldstein et al., 1989 supra;Hampel et aL, 1990 Nucleic Acid Rest 18, 299; and Hampel et al., EP0360257.

Hairpin ribozyme substrate complex comprises of two intermolecularhelices formed between the ribozyme and the target RNA (helix 1 andhelix 2). Length of helix 1 can be varied substantially withouteffecting the catalytic activity of the ribozyme (Hisamatsu et al., 1993supra). However, the length of helix 2 is reported to be sensitive tovariation. The length of helix 2 is normally between 3 and 5 base-pairslong (Hampel & Tritz, 1989 supra; Feldstein et al., 1989 supra; Haseloffand Gerlach, 1989 supra; Hampel et al., 1990 supra; Feldstein et al.,1990 Proc. Natl. Acad. Sci. USA 87, 2623). Several reports suggest thatmutations within this helix significantly inhibit ribozyme activity(Hampel et al., 1990 supra; Feldstein et al., 1990 supra; Chowrira &Burke, 1991 Biochemistry 30, 8518; Joseph et al., 1993 Genes & Develop.7, 130). It is also believed in the art that the length of helix 2should be between 3 and 5 bp (Hampel et al., 1988 EPO 360 257; Hampel etal., 1993 supra, Cech, 1992 supra; von Ahsen and Schroeder, 1993 supra;Hisamatsu et al., 1993 supra, Anderson et al., 1994 supra).

SUMMARY OF THE INVENTION

This invention features an improved trans-cleaving hairpin ribozyme inwhich a new helix (i.e., a sequence able to form a double-strandedregion with another single-stranded nucleic acid) is provided in theribozyme to base-pair with a 5' region of a separate substrate nucleicacid. This helix is provided at the 3' end of the ribozyme after helix 3as shown in FIG. 6. In addition, at least two extra bases may beprovided in helix 2 and a portion of the substrate corresponding tohelix 2 may be either directly linked to the 5' portion able to hydrogenbond to the 3' end of the hairpin or may have a linker of atleast onebase. By trans-cleaving is meant that the ribozyme is able to act intrans to cleave another RNA molecule which is not covalently linked tothe ribozyme itself. Thus, the ribozyme is not able to act on itself inan intramolecular cleavage reaction.

By "base-pair" is meant a nucleic acid that can form hydrogen bond(s)with other RNA sequence by either traditional Watson-Crick or othernon-traditional types (for example Hoogsteen type) of interactions.

Referring to FIG. 6, there is shown in diagrammatic form the generalstructure of a ribozyme of this invention. Helix 2 (H2) is provided witha least 4 base pairs (i.e., n is 1, 2, 3 or 4) and a new helix 5 isprovided of length 2 or more bases (preferably 3-20 bases, i.e., m isfrom 1-20 or more). Helix 2 and helix 5 may be covalently linked by oneor more bases (i.e., r is≧1 base). Together, these modifications of thehairpin (HP) structure are advantageous in enhancing stability to theribozyme-substrate complex. Helix 4 may also be extended by 2 or morebase pairs (e.g., 4-10 base pairs) to stabilize the ribozyme structure.In each instances, each N and N' independently is any normal or modifiedbase and each dash represents a potential base-pairing interaction.These nucleotides may be modified at the sugar, base or phosphate.Complete base-pairing is not required in the helixes, but is preferred.Helix 1 and 4 can be of any size (i.e., o and p is each independentlyfrom 0 to any number, e.g., 20) as long as some base-pairing ismaintained. Essential bases are shown as specific bases in thestructure, but those in the art will recognize that one or more may bemodified chemically (abasic, base, sugar and/or phosphate modifications)or replaced with another base without significant effect. Helix 4 can beformed from two separate molecules, i.e., without a connecting loop. Theconnecting loop when present may be a ribonucleotide with or withoutmodifications to its base, sugar or phosphate. "q" is ≧2 bases. Theconnecting loop can also be replaced with a non-nucleotide linkermolecule (see FIG. 17 for more details). H, refers to bases A, U or C. Yrefers to pyrimidine bases. "------" refers to a chemical bond.

The increase in length of helix 2 of a hairpin ribozyme (with or withouthelix 5) has several advantages. These include improved stability of theribozyme-target complex in vivo. In addition, an increase in therecognition sequence of the hairpin ribozyme improves the specificity ofthe ribozyme. This also makes possible the targeting of potentialhairpin ribozyme sites that would otherwise be inaccessible due toneighboring secondary structure.

The increase in length of helix 2 of a hairpin ribozyme (with or withouthelix 5) enhances trans-ligation reaction catalyzed by the ribozyme.Transligation reactions catalyzed by the regular hairpin ribozyme (4 bphelix 2) is very inefficient (Komatsu et al., 1993 Nucleic Acids Res.21, 185). This is attributed to weak base-pairing interactions betweensubstrate RNAs and the ribozyme. By increasing the length of helix 2(with or without helix 5) the rate of ligation (in vitro and in vivo)can be enhanced several fold.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims.

DESCRIPTION OF PREFERRED EMBODIMENTS

The drawings will first briefly be described.

Drawings:

FIG. 1 is a diagrammatic representation of the hammerhead ribozymedomain known in the art. Stem II can be ≧2 base-pair long, or can evenlack base pairs and consist of a loop region.

FIG. 2a is a diagrammatic representation of the hammerhead ribozymedomain known in the art;

FIG. 2b is a diagrammatic representation of the hammerhead ribozyme asdivided by Uhlenbeck (1987, Nature, 327, 596-600) into a substrate andenzyme portion;

FIG. 2c is a similar diagram showing the hammerhead divided by Haseloffand Gerlach (1988, Nature, 334, 585-591) into two portions; and

FIG. 2d is a similar diagram showing the hammerhead divided by Jeffriesand Symons (1989, Nucleic. Acids. Res., 17, 1371-1371) into twoportions.

FIG. 3 is a representation of the general structure of the hairpinribozyme domain known in the art. H, is A, U or C. Y is U or C. N is A,U, G, or C. N' is the complementary sequence of N. Helix 4 can be ≧2base-pair long.

FIG. 4 is a representation of the general structure of the hepatitisdelta virus ribozyme domain known in the art.

FIG. 5 is a representation of the general structure of the VS RNAribozyme domain.

FIG. 6 is a diagrammatic representation of a ribozyme of this invention(see above for a complete description).

FIG. 7 is a diagrammatic representation of a Site A Hairpin Ribozyme(HP-A) showing the proposed secondary structure of the hairpin ribozymesubstrate complex as described in the art (Berzal-Herranz et al., 1993EMBO. J. 12, 2567). The ribozyme has been assembled from two fragments(bimolecular ribozyme; Chowrira and Burke, 1992 Nucleic Acids Res. 20,2835); #H1 and H2 represent intermolecular helix formation between theribozyme and the substrate. H3 and H4 represent intramolecular helixformation within the ribozyme (intermolecular helix in the case ofbimolecular ribozyme). Left panel (HP-Al) indicates 4 base-paired helix2 and the right panel (HP-A2) indicates 6 base-paired helix 2. Arrowindicates the site of RNA cleavage. All the ribozymes discussed hereinwere chemically synthesized by solid phase synthesis using RNAphosphoramadite chemistry, unless otherwise indicated. Those skilled inthe art will recognize that these ribozymes could also be madetranscriptionally in vitro and in vivo.

FIG. 8 of a graph showing RNA cleavage by hairpin ribozymes targeted tosite A. A plot of fraction of the target RNA uncleaved (fractionuncleaved) as a function of time is shown. HP-A2 (6 bp helix 2) cleavesa 422 target RNA to a greater extent than the HP-A1 (4 bp helix 2).

To make internally-labeled substrate RNA for trans-ribozyme cleavagereactions, a 422 nt region (containing hairpin site A) was synthesizedby PCR using primers that place the T7 RNA promoter upstream of theamplified sequence. Target RNA was transcribed in a standardtranscription buffer in the presence of [α-³² P]CTP (Chowrira & Burke,1991 supra). The reaction mixture was treated with 15 units ofribonuclease-free DNasel, extracted with phenol followedchloroform:isoamyl alcohol (25:1), precipitated with isopropanol andwashed with 70% ethanol. The dried pellet was resuspended in 20 μlDEPC-treated water and stored at -20° C.

Unlabeled ribozyme (1 μM) and internally labeled 422 nt substrate RNA(<10 nM) were denatured and renatured separately in a standard cleavagebuffer (containing 50 mM Tris.HCl pH 7.5 and 10 mM MgCl₂) by heating to90° C. for 2 min. and slow cooling to 37° C. for 10 min. The reactionwas initiated by mixing the ribozyme and substrate mixtures andincubating at 37° C. Aliquots of 5 μl were taken at regular timeintervals, quenched by adding an equal volume of 2× formamide gelloading buffer and frozen on dry ice. The samples were resolved on 5%polyacrylamide sequencing gel and results were quantitatively analyzedby radioanalytic imaging of gels with a Phosphorlmager (MolecularDynamics, Sunnyvale, Calif.)

FIG. 9 is the Site B Hairpin Ribozyme (HP-B) showing proposed secondarystructure of the hairpin ribozyme substrate complex. The ribozyme wasassembled from two fragments as described above. The nomenclature is thesame as above.

FIG. 10 a graph showing RNA cleavage by hairpin ribozymes targeted tosite B. A. plot of fraction of the target RNA uncleaved (fractionuncleaved) as a function of time is shown. HP-B2 (6 bp helix 2) cleavesa 2 KB target RNA to a greater extent than the HP-B1 (4 bp helix 2). Tomake intemally-labeled substrate RNA for trans-ribozyme cleavagereactions, a 2 kB region (containing hairpin site B) was synthesized byPCR using primers that place the T7 RNA promoter upstream of theamplified sequence. The cleavage reactions were carried out as describedabove.

FIG. 11 shows a Site C Hairpin Ribozyme (HP-C) with the proposedsecondary structure of the hairpin ribozyme substrate complex. Theribozyme was assembled from two fragments as described above.

FIG. 12 is a graph showing RNA cleavage by hairpin ribozymes targeted tosite C. The ribozymes were tested at both 20° C. and at 26° C. To makeinternally-labeled substrate RNA for trans-ribozyme cleavage reactions,a 1.9 KB region (containing hairpin site C) was synthesized by PCR usingprimers that place the T7 RNA promoter upstream of the amplifiedsequence. Cleavage reactions were carried out as described above exceptthat 20° C. and at 26° C. temperatures were used.

FIGS. 13 and 15 show Site D and E hairpin ribozymes (HP-D/E) within aproposed secondary structure of hairpin ribozyme substrate complex. Twohairpin ribozymes were designed to cleave two distinct sites (sites Dand sites E) within the mouse TNF-α mRNA. Two more ribozymes weresynthesized containing mutations in the catalytic core of the ribozymewhich renders the ribozyme inactive (Berzal-Herranz et al., 1993 EMBO J.12, 2567). To construct these ribozymes, partially overlapping top- andbottom-strand oligonucleotides (˜50 nucleotides) were designed toinclude sequences for the T7 promoter and the hairpin ribozyme. Thesingle-strand portions of annealed oligonucleotides were converted todouble-strands using Sequenase® (U.S. Biochemicals). Transcriptionreactions containing linear double-stranded templates were carried outessentially as described (Milligan & Uhlenbeck, 1989 supra) using the T7mega shortscript kit (Ambion, Austin, Tex.).

FIGS. 14 and 16 show RNA cleavage by HP-D and HP-E in mammalian cellswith noted controls.

FIGS. 17A-17D shows various structural modifications of the presentinvention. A) Hairpin ribozyme lacking helix 5. Nomenclature is same asdescribed under FIG. 6. B) Hairpin ribozyme lacking helix 4 and helix 5.Helix 4 is replaced by a nucleotide loop wherein q is ≧2 bases.Nomenclature is same as described under FIG. 6. C) Hairpin ribozymelacking helix 5. Helix 4 loop is replaced by a linker "L", wherein L isa non-nucleotide linker molecule (Benseler et al., 1993 J. Am. Chem.Soc. 115, 8483; Jennings et al., WO 94/13688). Nomenclature is same asdescribed under FIG. 6. D) Hairpin ribozyme lacking helix 4 and helix 5.Helix 4 is replaced by non-nucleotide linker molecule "L" (Benseler etal., 1993 supra; Jennings et al., supra). Nomenclature is same asdescribed under FIG. 6.

FIGS. 18A and B show Hairpin ribozymes containing nucleotide spacerregion "s" at the indicated location, wherein s is ≧1 base. Hairpinribozymes containing spacer region, can be synthesized as one fragmentor can be assembled from multiple fragments. Nomenclature is same asdescribed under FIG. 6.

TARGET SITES

Targets for useful ribozymes can be determined as disclosed in Draper etal . WO 93/23569, Sullivan et al., WO 94/02595 as well as by Draper etal., "Method and reagent for treatment of arthritic conditions U.S. Ser.No. 08/152,487, filed Nov. 12, 1993, abandoned, and hereby incorporatedby reference herein in totality. Rather than repeat the guidanceprovided in those documents here, below are provided specific examples,not limiting to those in the art. Ribozymes to such targets are designedas described in those applications and synthesized to be tested in vitroand in vivo, as also described. Such ribozymes can also be optimized anddelivered as described therein.

Ribozymes are designed to anneal to various sites in the target RNA. Thebinding arms are complementary to the target site sequences describedabove. The ribozymes are chemically synthesized. The method of synthesisused follows the procedure for normal RNA synthesis as described inUsman et al., 1987 J. Am. Chem. Soc., 109, 7845-7854 and in Scaringe etal., 1990 Nucleic Acids Res., 18, 5433-5441 and makes use of commonnucleic acid protecting and coupling groups, such as dimethoxytrityl atthe 5'-end, and phosphoramidites at the 3'-end. The average stepwisecoupling yields were >98%. Hairpin ribozymes are synthesized in twoparts and annealed to reconstruct the active ribozyme (Chowrira andBurke, 1992 Nucleic Acids Res., 20, 2835-2840). All ribozymes aremodified extensively to enhance stability by modification with nucleaseresistant groups, for example, 2'-amino, 2'-C-allyl, 2'-flouro,2'-o-methyl, 2'-H (for a review see Usman and Gedergren, 1992 TIBS 17,34). Ribozymes are purified by gel electrophoresis using general methodsor are purified by high pressure liquid chromatography (HPLC; See Usmanet al., Synthesis, deprotection, analysis and purification of RNA andribozymes, filed May, 18, 1994, U.S. Ser. No. 08/245,736, abandoned, thetotality of which is hereby incorporated herein by reference) and areresuspended in water.

Ribozyme activity can be optimized by chemically synthesizing ribozymeswith modifications that prevent their degradation by serum ribonucleases(see e.g., Eckstein et al., International Publication No. WO 92/07065;Perrault et al., Nature 1990, 344:565; Pieken et al., Science 1991,253:314; Usman and Cedergren, Trends in Biochem. Sci. 1992, 17:334;Usman et al., International Publication No. WO 93/15187; and Rossi etal., International Publication No. WO 91/03162, as well as Usman, N. etal., U.S. patent application Ser. No. 07/829,729, U.S. Pat. No.5,652,094, and Sproat, B. European Patent Application 92110298.4;Ortigao et al., 2 Antisense research and Development; Krist et al.,Abstracts International conference on antisense nucleic acids,Garmisch-Partenkirchen, 1993; Chowrira and Burke, 1992 supra; Chowriraet al., 1993 J. BioL Chem. 268, 19458, which describe various chemicalmodifications that can be made to the sugar moieties of enzymatic RNAmolecules. All these publications are hereby incorporated by referenceherein.), modifications which enhance their efficacy in cells, andremoval of helix-containing bases to shorten RNA synthesis times andreduce chemical requirements.

Ribozymes are added directly, or can be complexed with cationic lipids,packaged within liposomes, or otherwise delivered to target cells. TheRNA or RNA complexes can be locally administered to relevant tissues exviva, or in vivo through injection, aerosol inhalation, infusion pump orstent, with or without their incorporation in biopolymers.

Sullivan, et al., supra, describes the general methods for delivery ofenzymatic RNA molecules. Ribozymes may be administered to cells by avariety of methods known to those familiar to the art, including, butnot restricted to, encapsulation in liposomes, by iontophoresis, or byincorporation into other vehicles, such as hydrogels, cyclodextrins,biodegradable nanocapsules, and bioadhesive microspheres. For someindications, ribozymes may be directly delivered ex vivo to cells ortissues with or without the aforementioned vehicles. Alternatively, theRNA/vehicle combination is locally delivered by direct injection or byuse of a catheter, infusion pump or stent. Other routes of deliveryinclude, but are not limited to, intravascular, intramuscular,subcutaneous or joint injection, aerosol inhalation, oral (tablet orpill form), topical, systemic, ocular, intraperitoneal and/orintrathecal delivery. More detailed descriptions of ribozyme deliveryand administration are provided in Sullivan, et al., supra and Draper,et al., supra which have been incorporated by reference herein.

Another means of accumulating high concentrations of a ribozyme(s)within cells is to incorporate the ribozyme-encoding sequences into aDNA expression vector. Transcription of the ribozyme sequences aredriven from a promoter for eukaryotic RNA polymerase I (pol I), RNApolymerase II (pol II), or RNA polymerase III (pol III). Transcriptsfrom pol II or pol III promoters will be expressed at high levels in allcells; the levels of a given pol II promoter in a given cell type willdepend on the nature of the gene regulatory sequences (enhancers,silencers, etc.) present nearby. Prokaryotic RNA polymerase promotersare also used, providing that the prokaryotic RNA polymerase enzyme isexpressed in the appropriate cells (Elroy-Stein, O. and Moss, B., 1990,Proc. Natl. Acad. Sci. U S A, 87, 6743-7; Gao, X. and Huang; L., 1993,Nucleic Acids Res., 21, 2867-72; Lieber, A., et al., 1993, MethodsEnzymol., 217, 47-66; Zhou, Y., et al., 1990, Mol. Cell. Biol., 10,4529-37). Several investigators have demonstrated that ribozymesexpressed from such promoters can function in mammalian cells (e.g.(Kashani-Sabet, M., et al., 1992, Antisense Res. Dev., 2, 3-15; Ojwang,J. O., et al., 1992, Proc. Natl. Acad. Sci. U S A, 89, 10802-6; Chen, C.J., et al., 1992, Nucleic Acids Res., 20, 4581-9; Yu, M., et al., 1993,Proc. Natl. Acad. Sci. U S A, 90, 6340-4; L'Huillier, P. J., et al.,19921, EMBO J., 11, 4411-8; Lisziewicz, J., et a., 1993, Proc. Natl.Acad. Sci. U. S. A., 90, 8000-4)). The above ribozyme transcriptionunits can be incorporated into a variety of vectors for introductioninto mammalian cells, including but not restricted to, plasmid DNAvectors, viral DNA vectors (such as adenovirus or adeno-associatedvectors), or viral RNA vectors (such as retroviral, Semliki forestvirus, sindbis virus vectors).

In a preferred embodiment of the invention, a transcription unitexpressing a hairpin ribozyme that cleaves target RNA (e.g., TNF-α mRNA)is inserted into a plasmid DNA vector or an adenovirus oradeno-associated DNA viral vector. Both viral vectors have been used totransfer genes to the lung and both vectors lead to transient geneexpression (Zabner et al., 1993 Cell 75, 207; Carter, 1992 Curr. Opi.Biotech. 3, 533). The adenovirus vector is delivered as recombinantadenoviral particles. DNA may be delivered alone or complexed withvehicles (as described for RNA above). The DNA, DNA/vehicle complexes,or the recombinant adenovirus particles are locally administered to thesite of treatment, e.g., through the use of an injection catheter, stentor infusion pump or are directly added to cells or tissues ex vivo.

In another aspect of the invention, ribozymes that cleave targetmolecules are expressed from transcription units inserted into DNA, RNA,or viral vectors. Preferably, the recombinant vectors capable ofexpressing the ribozymes are locally delivered as described above, andtransiently persist in target cells. Once expressed, the ribozymescleave the target mRNA. The recombinant vectors are preferably DNAplasmids, adenovirus, retroviral or adeno-associated virus vectors.However, other mammalian cell vectors that direct the expression of RNAmay be used for this purpose.

Thus, ribozymes of the present invention that cleave target mRNA andthereby inhibit and/or reduce target activity have many potentialtherapeutic uses, and there are reasonable modes of delivering theribozymes in a number of the possible indications. Development of aneffective ribozyme that inhibits specific function are described in theart.

By "Inhibit" is meant that the activity or level of target RNA isreduced below that observed in the absence of the ribozyme, andpreferably is below that level observed in the presence of an inactiveRNA molecule able to bind to the same site on theRNA, but unable tocleave that RNA.

By "vectors" is meant any nucleic acid- and/or viral-based techniqueused to deliver a desired nucleic acid.

Diagnostic uses

Ribozymes of this invention may be used as diagnostic tools to examinegenetic drift and mutations within diseased cells, or to detect specificRNA molecules, such as virus RNA. The close relationship betweenribozyme activity and the structure of the target RNA allows thedetection of mutations in any region of the molecule which alters thebase-pairing and three-dimensional structure of the target RNA. By usingmultiple ribozymes described in this invention, one may map nucleotidechanges which are important to RNA structure and function in vitro, aswell as in cells and tissues. Cleavage of target RNAs with ribozymes maybe used to inhibit gene expression and define the role (essentially) ofspecified gene products in the progression of disease. In this manner,other genetic targets may be defined as important mediators of thedisease. These experiments will lead to better treatment of the diseaseprogression by affording the possibility of combinational therapies(e.g., multiple ribozymes targeted to different genes, ribozymes coupledwith known small molecule inhibitors, or intermittent treatment withcombinations of ribozymes and/or other chemical or biologicalmolecules). Other in vitro uses of ribozymes of this invention are wellknown in the art, and include detection of the presence of mRNAassociated with a related condition. Such RNA is detected by determiningthe presence of a cleavage product after treatment with a ribozyme usingstandard methodology.

In a specific example, ribozymes which can cleave only wild-type ormutant forms of the target RNA are used for the assay. The firstribozyme is used to identify wild-type RNA present in the sample and thesecond ribozyme will be used to identify mutant RNA in the sample. Asreaction controls, synthetic substrates of both wild-type and mutant RNAwill be cleaved by both ribozymes to demonstrate the relative ribozymeefficiencies in the reactions and the absence of cleavage of the"non-targeted" RNA species. The cleavage products from the syntheticsubstrates will also serve to generate size markers for the analysis ofwild-type and mutant RNAs in the sample population. Thus each analysiswill require two ribozymes, two substrates and one unknown sample whichwill be combined into six reactions. The presence of cleavage productswill be determined using an RNAse protection assay so that full-lengthand cleavage fragments of each RNA can be analyzed in one lane of apolyacrylamide gel. It is not absolutely required to quantify theresults to gain insight into the expression of mutant RNAs and putativerisk of the desired phenotypic changes in target cells. The expressionof mRNA whose protein product is implicated in the development of thephenotype is adequate to establish risk. If probes of comparablespecific activity are used for both transcripts, then a qualitativecomparison of RNA levels will be adequate and will decrease the cost ofthe initial diagnosis. Higher mutant form to wild-type ratios will becorrelated with higher risk whether RNA levels are comparedqualitatively or quantitatively.

EXAMPLES

Results of experiments suggest that the length of H2 can be 6 bp withoutsignificantly reducing the activity of the hairpin ribozyme. The H2 armlength variation does not appear to be sequence dependent. HP ribozymeswith 6 bp H2 have been designed against five different target RNAs andall five ribozymes efficiently cleaved their cognate target RNA.Additionally, two of these ribozymes were able to successfully inhibitgene expression (e.g., TNF-α) in mammalian cells. Results of theseexperiments are shown below.

HP ribozymes with 7 and 8 bp H2 are also capable of cleaving target RNAin a sequence-specific manner, however, the rate of the cleavagereaction is lower than those catalyzed by HP ribozymes with 6 bp H2.

Example 1: 4 and 6 base pair H2

Referring to FIGS. 7-12, HP ribozymes were synthesized as describedabove and tested for activity. Surprisingly, those with 6 base pairs inH2 were still as active as those with 4 base pairs.

Example 2: TNF α ribozymes

Referring to FIGS. 13-16, hairpin ribozymes of this invention weresynthesized as described, and tested in a mammalian cell system asdescribed below.

Macrophage isolation. To produce responsive macrophages 1 ml of sterilefluid thioglycollate broth (Difco, Detroit, Mich.) was injected i.p.into 6 week old female C57bI/6NCRmice 3 days before peritoneal lavage.Mice were maintained as specific pathogen free in autoclaved cages in alaminar flow hood and given sterilized water to minimize "spontaneous"activation of macrophages. The resulting peritoneal exudate cells (PEC)were obtained by lavage using Hanks balanced salt solution (HBSS),plated at 2.5×10⁵ /well in 96 well plates (Costar, Cambridge, Mass.)with Eagles minimal essential medium (EMEM) with 10% heat inactivatedfetal bovine serum. After adhering for 2 hours the wells were washed toremove non adherent cells. The resulting cultures were 97% macrophagesas determined by morphology and staining for non-specific esterase.

Transfection of ribozymes into macrophages: The ribozymes were dilutedto 2× final concentration, mixed with an equal volume of 11 nMlipofectamine (Life Technologies, Gaithersburg, Md.), and vortexed. 100ml of lipid:ribozyme complex was then added directly to the cells,followed immediately by 10 ml fetal bovine serum. Three hours afterribozyme addition 100 ml of 1 mg/ml bacterial lipopolysaccaride (LPS)was added to each well to stimulate TNF production.

Quantitation of TNF-α in mouse macrophages: Supernatants were sampled at0, 2, 4, 8, and 24 hours post LPS stimulation and stored at -70° C.until quantitation, which was done by a TNF-α specific ELISA. ELISAplates were coated with rabbit anti-mouse TNF-a serum at 1;1000 dilution(Genzyme) followed by blocking with blotto and incubation with TNF-αcontaining supematants. TNF-α was then detected using a murine TNF-αspecific hamster monoclonal antibody (Genzyme). The ELISA was developedwith goat anti-hamster IgG coupled to alkaline phosphatase.

Assessment of reagent toxicity: Following ribozymellipid treatment ofmacrophages and harvesting of supernatants viability of the cells wasassessed by incubation of the cells with 5 mg/ml of3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT).This compound is reduced by the mitochondrial dehydrogenases, theactivity of which correlates well with cell viability. After 12 hoursthe absorbance of reduced MTT is measured at 585 nm.

Inhibition of TNF-α expression by hairpin ribozymes in mousemacrophages: As shown in FIGS. 14 and 16, expression of TNF-α issignificantly inhibited by both active hairpin ribozymes HP-D and HP-E.

The inhibition of TNF-α expression appears to be dependent on thecatalytic activity of the ribozyme, because, catalytically inactivehairpin ribozyme (HP-D dead) does not show appreciable inhibition. HP-E(inactive) ribozyme does show some inhibition of TNF-α expression at 162nM ribozyme concentration. This inhibition may be attributed to someantisense effect.

Other embodiments are within the following claims.

                  TABLE I                                                         ______________________________________                                        Characteristics of Ribozymes                                                  Group I Introns                                                                Size: -200 to >1000 nucleotides.                                              Requires a U in the target sequence immediately 5' of the cleavage            site.                                                                         Binds 4-6 nucleotides at 5' side of cleavage site.                            Over 75 known members of this class. Found in Tetrahymena                     thermophila rRNA, fungal mitochondria, chloroplasts, phage T4,                blue-green algae, and others.                                                 RNAseP RNA (M1 RNA)                                                           Size: -290 to 400 nucleotides.                                                RNA portion of a ribonucleoprotein enzyme. Cleaves tRNA                       precursors to form mature tRNA.                                               Roughly 10 known members of this group all are bacterial in origin.           Hammerhead Ribozyme                                                           Size: -13 to 40 nucleotides.                                                  Requires the target sequence UH immediately 5' of the cleavage                site.                                                                         Binds a variable number nucleotides on both sides of the cleavage             site.                                                                         14 known members of this class. Found in a number of plant                    pathogens (virusoids) that use RNA as the infectious agent (FIG.              1)                                                                            Hairpin Ribozyme                                                              Size: -50 nucleotides.                                                        Requires the target sequence GUC immediately 3' of the cleavage               site.                                                                         Binds 4-6 nucleotides at 5' side of the cleavage site and a variable          number to the 3' side of the cleavage site.                                   Only 3 known member of this class. Found in three plant pathogen              (satellite RNAs of the tobacco ringspot virus, arabis mosaic virus            and chicory yellow mottle virus) which uses RNA as the infectious             agent (FIG. 3).                                                               Hepatitis Delta Virus (HDV) Ribozyme                                          Size: 50-60 nucleotides (at present).                                         Cleavage of target RNAs recently demonstrated.                                Sequence requirements not fully determined.                                   Binding sites and structural requirements not fully determined,               although no sequences 5' of cleavage site are required.                       Only 1 known member of this class. Found in human HDV (FIG.                   4).                                                                           Neuraspora VS RNA Ribozyme                                                    Size: -144 nucleotides (at present)                                           Cleavage of target RNAs recently demonstrated.                                Sequence requirements not fully determined.                                   Binding sites and structural requirements not fully determined. Only          1 known member of this class. Found in Neuraspara VS RNA                      (FIG. 5).                                                                    ______________________________________                                    

    __________________________________________________________________________    #             SEQUENCE LISTING                                                  - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES:       - #         48                        - -  - - (2) INFORMATION FOR SEQ ID NO:    1:                                 - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #           11 base pairs                                   (B) TYPE:     - #             nucleic acid                                    (C) STRANDEDNESS:   - #       single                                          (D) TOPOLOGY:    - #          linear                                 - -     (ix) FEATURE:                                                                  (D) OTHER INFORMATION: - #  The letter "N" stands for any          base.                                                                                          The lette - #r "H" stands for C, A or U.                        - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  1:                        - - NNNNUHNNNN N               - #                  - #                      - #       11                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:    2:                                 - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #           28 base pairs                                   (B) TYPE:     - #             nucleic acid                                    (C) STRANDEDNESS:   - #       single                                          (D) TOPOLOGY:    - #          linear                                 - -     (ix) FEATURE:                                                                  (D) OTHER INFORMATION: - # The letter "N" stands for any base.       - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  2:                        - - NNNNNCUGAN GAGNNNNNNC GAAANNNN         - #                  - #                 28                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 3:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #           14 base pairs                                   (B) TYPE:     - #             nucleic acid                                    (C) STRANDEDNESS:   - #       single                                          (D) TOPOLOGY:    - #          linear                                 - -     (ix) FEATURE:                                                                  (D) OTHER INFORMATION: - #  The letter "N" stands for any          base.                                                                                          The lette - #r "Y" is stands for U or C.                                      The lette - #r "H" stands for A, U or C.                        - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #3:                           - - NNNYNGHYNN NNNN              - #                  - #                      - #     14                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO: 4:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #           50 base pairs                                   (B) TYPE:     - #             nucleic acid                                    (C) STRANDEDNESS:   - #       single                                          (D) TOPOLOGY:    - #          linear                                 - -     (ix) FEATURE:                                                                  (D) OTHER INFORMATION: - # The letter "N" stands for any base.       - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - NNNNNNNGAA GNNNNNNNNN NAAACANNNN NNNNNNNACA UUACNNNNNN  - #                  50                                                                         - -  - - (2) INFORMATION FOR SEQ ID NO:    5:                                 - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #           85 base pairs                                   (B) TYPE:     - #             nucleic acid                                    (C) STRANDEDNESS:   - #       single                                          (D) TOPOLOGY:    - #          linear                                 - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  5:                        - - UGGCCGGCAU GGUCCCAGCC UCCUCGCUGG CGCCGGCUGG GCAACAUUCC GA -             #GGGGACCG     60                                                                 - - UCCCCUCGGU AAUGGCGAAU GGGAC          - #                  - #                   85                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:    6:                                 - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #           176 base pairs                                  (B) TYPE:     - #             nucleic acid                                    (C) STRANDEDNESS:   - #       single                                          (D) TOPOLOGY:    - #          linear                                 - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  6:                        - - GGGAAAGCUU GCGAAGGGCG UCGUCGCCCC GAGCGGUAGU AAGCAGGGAA CU -             #CACCUCCA     60                                                                 - - AUUUCAGUAC UGAAAUUGUC GUAGCAGUUG ACUACUGUUA UGUGAUUGGU AG -            #AGGCUAAG    120                                                                 - - UGACGGUAUU GGCGUAAGUC AGUAUUGCAG CACAGCACAA GCCCGCUUGC GA - #GAAU            176                                                                       - - (2) INFORMATION FOR SEQ ID NO:    7:                                      - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #           15 base pairs                                   (B) TYPE:     - #             nucleic acid                                    (C) STRANDEDNESS:   - #       single                                          (D) TOPOLOGY:    - #          linear                                 - -     (ix) FEATURE:                                                                  (D) OTHER INFORMATION: - #  The letter "N" stands for any          base.                                                                                          The lette - #r "Y" is stands for U or C.                                      The lette - #r "H" stands for A, U or C.                        - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  7:                        - - NNNNNNNYNG HYNNN              - #                  - #                      - #    15                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:    8:                                 - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #           47 base pairs                                   (B) TYPE:     - #             nucleic acid                                    (C) STRANDEDNESS:   - #       single                                          (D) TOPOLOGY:    - #          linear                                 - -     (ix) FEATURE:                                                                  (D) OTHER INFORMATION: - #  The letter "N" stands for any          base.                                                                                          The lette - #r "H" stands for C, A or U.                        - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  8:                        - - NNNNGAAGNN NNNNNNNNNA AAHANNNNNN NACAUUACNN NNNNNNN   - #                    47                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO: 9:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #           14 base pairs                                   (B) TYPE:     - #             nucleic acid                                    (C) STRANDEDNESS:   - #       single                                          (D) TOPOLOGY:    - #          linear                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #9:                           - - AUACGGUCCC CUGA              - #                  - #                      - #     14                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 10:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #           32 base pairs                                   (B) TYPE:     - #             nucleic acid                                    (C) STRANDEDNESS:   - #       single                                          (D) TOPOLOGY:    - #          linear                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #10:                          - - UCAGGGAGAA GUAUACCAGA GAAACACACG CG       - #                  - #              32                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 11:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #           21 base pairs                                   (B) TYPE:     - #             nucleic acid                                    (C) STRANDEDNESS:   - #       single                                          (D) TOPOLOGY:    - #          linear                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #11:                          - - CGCGUGGUAC AUUACCUGGU A           - #                  - #                      - #21                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 12:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #           16 base pairs                                   (B) TYPE:     - #             nucleic acid                                    (C) STRANDEDNESS:   - #       single                                          (D) TOPOLOGY:    - #          linear                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #12:                          - - AAAUACGGUC CCCUGA             - #                  - #                      - #    16                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 13:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #           34 base pairs                                   (B) TYPE:     - #             nucleic acid                                    (C) STRANDEDNESS:   - #       single                                          (D) TOPOLOGY:    - #          linear                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #13:                          - - UCAGGGAGAA GUAUUUACCA GAGAAACACA CGCG       - #                  -      #        34                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 14:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #           21 base pairs                                   (B) TYPE:     - #             nucleic acid                                    (C) STRANDEDNESS:   - #       single                                          (D) TOPOLOGY:    - #          linear                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #14:                          - - CGCGUGGUAC AUUACCUGGU A           - #                  - #                      - #21                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO: 15:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #           16 base pairs                                   (B) TYPE:     - #             nucleic acid                                    (C) STRANDEDNESS:   - #       single                                          (D) TOPOLOGY:    - #          linear                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #15:                          - - CCGCCGUCUC CACCAA             - #                  - #                      - #    16                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 16:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #           34 base pairs                                   (B) TYPE:     - #             nucleic acid                                    (C) STRANDEDNESS:   - #       single                                          (D) TOPOLOGY:    - #          linear                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #16:                          - - UUGGUGGAAG AAGCGGACCA GAGAAACACA CGCG       - #                  -      #        34                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 17:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #           21 base pairs                                   (B) TYPE:     - #             nucleic acid                                    (C) STRANDEDNESS:   - #       single                                          (D) TOPOLOGY:    - #          linear                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #17:                          - - CGCGUGGUAC AUUACCUGGU A           - #                  - #                      - #21                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO: 18:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #           18 base pairs                                   (B) TYPE:     - #             nucleic acid                                    (C) STRANDEDNESS:   - #       single                                          (D) TOPOLOGY:    - #          linear                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #18:                          - - GUCCGCCGUC UCCACCAA             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 19:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #           36 base pairs                                   (B) TYPE:     - #             nucleic acid                                    (C) STRANDEDNESS:   - #       single                                          (D) TOPOLOGY:    - #          linear                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #19:                          - - UUGGUGGAAG AAGCGGACAC CAGAGAAACA CACGCG      - #                  -     #       36                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 20:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #           22 base pairs                                   (B) TYPE:     - #             nucleic acid                                    (C) STRANDEDNESS:   - #       single                                          (D) TOPOLOGY:    - #          linear                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #20:                          - - CGCGTUGGUA CAUUACCUGG UA           - #                  - #                     22                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 21:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #           18 base pairs                                   (B) TYPE:     - #             nucleic acid                                    (C) STRANDEDNESS:   - #       single                                          (D) TOPOLOGY:    - #          linear                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #21:                          - - CUGAGCUGUC CCCACCUG             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 22:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #           54 base pairs                                   (B) TYPE:     - #             nucleic acid                                    (C) STRANDEDNESS:   - #       single                                          (D) TOPOLOGY:    - #          linear                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #22:                          - - CAGGUGGGAG AAGCUCAGAC CAGAGAAACA CACGUUGUGG UACAUUACCU GG - #UA               54                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO: 23:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #           18 base pairs                                   (B) TYPE:     - #             nucleic acid                                    (C) STRANDEDNESS:   - #       single                                          (D) TOPOLOGY:    - #          linear                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #23:                          - - CUGAGCUGUC CCCACCUG             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 24:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #           54 base pairs                                   (B) TYPE:     - #             nucleic acid                                    (C) STRANDEDNESS:   - #       single                                          (D) TOPOLOGY:    - #          linear                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #24:                          - - CAGGUGGGAU AAGCUCAGAC CAGAGAAAGA CACGUUGUGG UACAUUACCU GG - #UA               54                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO: 25:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #           18 base pairs                                   (B) TYPE:     - #             nucleic acid                                    (C) STRANDEDNESS:   - #       single                                          (D) TOPOLOGY:    - #          linear                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #25:                          - - GGAAGCUGUC UUCAGACA             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 26:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #           54 base pairs                                   (B) TYPE:     - #             nucleic acid                                    (C) STRANDEDNESS:   - #       single                                          (D) TOPOLOGY:    - #          linear                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #26:                          - - UGUCUGAAAG AAGCUUCCAC CAGAGAAACA CACGUUGUGG UACAUUACCU GG - #UA               54                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO: 27:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #           18 base pairs                                   (B) TYPE:     - #             nucleic acid                                    (C) STRANDEDNESS:   - #       single                                          (D) TOPOLOGY:    - #          linear                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #27:                          - - GGAAGCUGUC UUCAGACA             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 28:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #           54 base pairs                                   (B) TYPE:     - #             nucleic acid                                    (C) STRANDEDNESS:   - #       single                                          (D) TOPOLOGY:    - #          linear                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #28:                          - - UGUCUGAAAU AAGCUUCCAC CAGAGAAAGA CACGUUGUGG UACAUUACCU GG - #UA               54                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO: 29:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #           11 base pairs                                   (B) TYPE:     - #             nucleic acid                                    (C) STRANDEDNESS:   - #       single                                          (D) TOPOLOGY:    - #          linear                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #29:                          - - NNNYNGHYNN N               - #                  - #                      - #       11                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 30:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #           43 base pairs                                   (B) TYPE:     - #             nucleic acid                                    (C) STRANDEDNESS:   - #       single                                          (D) TOPOLOGY:    - #          linear                                 - -     (ix) FEATURE:                                                                  (D) OTHER INFORMATION: - #  The letter "N" stands for any          base.                                                                                          The lette - #r "H" stands for C, A or U.                        - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #30:                          - - NNNNGAAGNN NNNNNNNNAA AHANNNNNNN ACAUUACNNN NNN    - #                      - # 43                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO: 31:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #           11 base pairs                                   (B) TYPE:     - #             nucleic acid                                    (C) STRANDEDNESS:   - #       single                                          (D) TOPOLOGY:    - #          linear                                 - -     (ix) FEATURE:                                                                  (D) OTHER INFORMATION: - #  The letter "N" stands for any          base.                                                                                          The lette - #r "Y" is stands for U or C.                                      The lette - #r "H" stands for A, U or C.                        - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO: 31:                      - -  NNNYNGHYNN N              - #                  - #                      - #       11                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO: 32:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #           39 base pairs                                   (B) TYPE:     - #             nucleic acid                                    (C) STRANDEDNESS:   - #       single                                          (D) TOPOLOGY:    - #          linear                                 - -     (ix) FEATURE:                                                                  (D) OTHER INFORMATION: - #  The letter "N" stands for any          base.                                                                                          The lette - #r "H" stands for C, A or U.                        - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #32:                          - - NNNNGAAGNN NNNNNNNNAA AHANNNACAU UACNNNNNN      - #                      - #    39                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO: 33:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #           11 base pairs                                   (B) TYPE:     - #             nucleic acid                                    (C) STRANDEDNESS:   - #       single                                          (D) TOPOLOGY:    - #          linear                                 - -     (ix) FEATURE:                                                                  (D) OTHER INFORMATION: - #  The letter "N" stands for any          base.                                                                                          The lette - #r "Y" is stands for U or C.                                      The lette - #r "H" stands for A, U or C.                        - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #33:                          - - NNNYNGHYNN N               - #                  - #                      - #       11                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO: 34:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #           25 base pairs                                   (B) TYPE:     - #             nucleic acid                                    (C) STRANDEDNESS:   - #       single                                          (D) TOPOLOGY:    - #          linear                                 - -     (ix) FEATURE:                                                                  (D) OTHER INFORMATION: - #  The letter "N" stands for any          base.                                                                                          The lette - #r "H" stands for C, A or U.                        - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #34:                          - - NNNNGAAGNN NNNNNNNNAA AHANN          - #                  - #                   25                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO: 35:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #           17 base pairs                                   (B) TYPE:     - #             nucleic acid                                    (C) STRANDEDNESS:   - #       single                                          (D) TOPOLOGY:    - #          linear                                 - -     (ix) FEATURE:                                                                  (D) OTHER INFORMATION: - # The letter "N" stands for any base.       - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #35:                          - - NNNNACAUUA CNNNNNN             - #                  - #                      - #   17                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 36:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #           11 base pairs                                   (B) TYPE:     - #             nucleic acid                                    (C) STRANDEDNESS:   - #       single                                          (D) TOPOLOGY:    - #          linear                                 - -     (ix) FEATURE:                                                                  (D) OTHER INFORMATION: - #  The letter "N" stands for any          base.                                                                                          The lette - #r "Y" is stands for U or C.                                      The lette - #r "H" stands for A, U or C.                        - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #36:                          - - NNNYNGHYNN N               - #                  - #                      - #       11                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO: 37:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #           23 base pairs                                   (B) TYPE:     - #             nucleic acid                                    (C) STRANDEDNESS:   - #       single                                          (D) TOPOLOGY:    - #          linear                                 - -     (ix) FEATURE:                                                                  (D) OTHER INFORMATION: - #  The letter "N" stands for any          base.                                                                                          The lette - #r "H" stands for C, A or U.                        - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #37:                          - - NNNNGAAGNN NNNNNNNNAA AHA           - #                  - #                    23                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO: 38:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #           15 base pairs                                   (B) TYPE:     - #             nucleic acid                                    (C) STRANDEDNESS:   - #       single                                          (D) TOPOLOGY:    - #          linear                                 - -     (ix) FEATURE:                                                                  (D) OTHER INFORMATION: - # The letter "N" stands for any base.       - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #38:                          - - NNACAUUACN NNNNN              - #                  - #                      - #    15                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 39:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #           15 base pairs                                   (B) TYPE:     - #             nucleic acid                                    (C) STRANDEDNESS:   - #       single                                          (D) TOPOLOGY:    - #          linear                                 - -     (ix) FEATURE:                                                                  (D) OTHER INFORMATION: - #  The letter "N" stands for any          base.                                                                                          The lette - #r "Y" is stands for U or C.                                      The lette - #r "H" stands for A, U or C.                        - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #39:                          - - NNNNNNNYNG HYNNN              - #                  - #                      - #    15                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO: 40:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #           47 base pairs                                   (B) TYPE:     - #             nucleic acid                                    (C) STRANDEDNESS:   - #       single                                          (D) TOPOLOGY:    - #          linear                                 - -     (ix) FEATURE:                                                                  (D) OTHER INFORMATION: - #  The letter "N" stands for any          base.                                                                                          The lette - #r "H" stands for C, A or U.                        - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #40:                          - - NNNNGAAGNN NNNNNNNNNA AAHANNNNNN NACAUUACNN NNNNNNN   - #                    47                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO: 41:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #           11 base pairs                                   (B) TYPE:     - #             nucleic acid                                    (C) STRANDEDNESS:   - #       single                                          (D) TOPOLOGY:    - #          linear                                 - -     (ix) FEATURE:                                                                  (D) OTHER INFORMATION: - #  The letter "N" stands for any          base.                                                                                          The lette - #r "Y" is stands for U or C.                                      The lette - #r "H" stands for A, U or C.                        - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #41:                          - - NNNYNGHYNN N               - #                  - #                      - #       11                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO: 42:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #           45 base pairs                                   (B) TYPE:     - #             nucleic acid                                    (C) STRANDEDNESS:   - #       single                                          (D) TOPOLOGY:    - #          linear                                 - -     (ix) FEATURE:                                                                  (D) OTHER INFORMATION: - #  The letter "N" stands for any          base.                                                                                          The lette - #r "H" stands for C, A or U.                        - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #42:                          - - NNNNGAAGNN NNNNNNNNNA AAHANNNNNN NACAUUACNN NNNNN   - #                      - #45                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO: 43:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #           20 base pairs                                   (B) TYPE:     - #             nucleic acid                                    (C) STRANDEDNESS:   - #       single                                          (D) TOPOLOGY:    - #          linear                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #43:                          - - CUGUAGUCCA GGAAUAUGGC            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 44:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #           38 base pairs                                   (B) TYPE:     - #             nucleic acid                                    (C) STRANDEDNESS:   - #       single                                          (D) TOPOLOGY:    - #          linear                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #44:                          - - GCCAUAUUCC UGAGAAGCAG ACCAGAGAAA CACACGCG      - #                      - #     38                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 45:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #           21 base pairs                                   (B) TYPE:     - #             nucleic acid                                    (C) STRANDEDNESS:   - #       single                                          (D) TOPOLOGY:    - #          linear                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #45:                          - - CGCGUGGUAC AUUACCUGGU A           - #                  - #                      - #21                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 46:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #           22 base pairs                                   (B) TYPE:     - #             nucleic acid                                    (C) STRANDEDNESS:   - #       single                                          (D) TOPOLOGY:    - #          linear                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #46:                          - - GACUGUAGUC CAGGAAUAUG GC           - #                  - #                     22                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 47:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #           40 base pairs                                   (B) TYPE:     - #             nucleic acid                                    (C) STRANDEDNESS:   - #       single                                          (D) TOPOLOGY:    - #          linear                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #47:                          - - GCCAUAUUCC UGAGAAGCAG UCACCAGAGA AACACACGCG     - #                      - #    40                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 48:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #           21 base pairs                                   (B) TYPE:     - #             nucleic acid                                    (C) STRANDEDNESS:   - #       single                                          (D) TOPOLOGY:    - #          linear                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #48:                          - - CGCGUGGUAC AUUACCUGGU A           - #                  - #                      - #21                                                                 __________________________________________________________________________

We claim:
 1. A method of cleaving a separate RNA molecule comprising thestep of contacting said separate RNA with a hairpin ribozyme comprisingribonucleotides, under conditions suitable for said cleaving, whereinsaid hairpin ribozyme comprises:(a) a helix 1 region comprising greaterthan or equal to three nucleotides inclusive, wherein said helix 1region is complementary to a region of said separate RNA molecule; (b) ahelix 2 region comprising between six and eight nucleotides inclusive,wherein said helix 2 region is complementary to a region of saidseparate RNA molecule; (c) a helix 3 region comprising nucleotides ableto form at least four base-pairs; and (d) a helix 4 region comprisingnucleotides able to form at least two base-pairs.
 2. The method of claim1, wherein said hairpin ribozyme comprises six nucleotides in the helix2 region.
 3. The method of claim 1, wherein said ribozyme has thefollowing structure: ##STR1## wherein each N and N' is independently anynucleotide; each A, U, G and C, is adenosine, uridine, guanosine andcytidine respectively and each Y is a pyrimidine nucleotide;each dashmay represent a base-pair; p is 1-20, q is 2-20 when present, o is 1-20,n is 3-5; and the lines at the 3' and the 5'-ends of the ribozyme aremeant to indicate additional nucleotides which may be present or absent.4. The method of claim 1, wherein said hairpin ribozyme has thefollowing structure: ##STR2## wherein each N and N' is independently anynucleotide; each A, U, G and C, is adenosine, uridine, guanosine andcytidine respectively and each Y is a pyrimidine nucleotide;each dashmay represent a base-pair; p is 1-20, o is 1-20, n is 3-5; L is anon-nucleotide linker; and the lines at the 3' and the 5'-ends of theribozyme are meant to indicate additional nucleotides which may bepresent or absent.
 5. The method of claim 1, wherein said hairpinribozyme comprises at least one sugar modification.
 6. The method ofclaim 1, wherein said hairpin ribozyme comprises at least one nucleotidebase modification.
 7. The method of claim 1, wherein said hairpinribozyme comprises at least one phosphate backbone modification.
 8. Themethod of claim 1, wherein said hairpin ribozyme is chemicallysynthesized.
 9. The method of claim 1, wherein said cleaving of saidseparate RNA molecule occurs in the presence of Mg² ".
 10. The method ofclaim 1, wherein said contacting of the separate RNA molecule with thehairpin ribozyme is in vitro.
 11. The method of claim 1, wherein saidcontacting of the separate RNA molecule with the hairpin ribozyme is ina mammalian cell.
 12. The method of claim 11, wherein said mammaliancell is a human cell.